Most tumour cells secrete certain growth factors to promote their proliferative activity. Our previous studies (disclosed in U.S. Pat. No. 6,156,725 and Australian Patent No. 707,158) have shown that peptides such as vasoactive intestinal peptide (VIP), somatostatin, bombesin and substance P also have specific high affinity receptors on the tumour cells, to which these peptides bind and which play a key role in cell proliferation. Specially designed analogs to these peptides can abrogate the proliferative effects of these peptides by binding to their receptors. It has also been shown from our studies (U.S. patent application Ser. No. 09/896,903) that when these peptides analogs are used in combination, they exert a synergistic effect, which is greater than when these analogs are used alone.
In our present invention novel conformationally constrained analogs of the above peptides (described in our U.S. Pat. No. 6,316,414 and U.S. patent application Ser. Nos. 09/630,333; 09/630,345 and 09/629,642) are linked together using suitable linkers consisting of pairs of basic amino acids to form a polypeptide. The individual analogs are released from the polypeptide by specific enzymatic cleavage at the linker site in vivo.
Such cleavage at pairs of basic residues is also known in biological systems. Most polypeptide hormones are synthesized as pro-hormones, and undergo endoproteolytic cleavage at pairs of basic residues (Lys-Arg, Arg-Arg) by enzymes called Prohormone Convertases (PCs), to give rise to the active molecule (Wilson, H. E and White, A, Trends Endocrinol. Metab. 1998, 9, 396-402; Jean F et al., Biochem. J. 1993, 292, 891-900, Mitra, J et al, Biochem J. 1998, 334, 275-282).
Of the bioactive peptides mentioned in the invention, Bombesin, is an amphibian peptide that has a structure closely related to that of several mammalian peptides, including Gastrin Releasing Peptide (GRP) and Neuromedins B and C. Bombesin, GRP and related peptides exert their in vivo effects by binding to specific receptors on cells of the gastrointestinal tract, the central nervous system and tumours. It has been reported (J. H. Walsh and J. R. Reeve, Peptides 6, (3), 63-68, (1985) that bombesin and bombesin-like peptides such as gastrin releasing peptide (GRP) are secreted by human small-cell lung cancer (SCLC) cells. It has been postulated (P. J. Woll and E. Rozengurt, PNAS 85, 1859-1863, (1988)) that gastrin releasing factor antagonists would bind competitively to bombesin receptors in animals and would therefore be of use in the treatment of SCLC and/or in the control of clinical symptoms associated with this disease and due to hypersecretion of this peptide hormone. Analogues of bombesin/GRP have been shown to inhibit the binding of gastrin releasing peptide to a SCLC cell line and to inhibit the growth of SCLC cells in-vitro and in-vivo (S. Mahmoud et al., CancerResearch, 1991, 51, 1798; Moody T W et al., Life Sci., 1995, 56, 521; Moody T W et al., Peptides, 1996, 17, 1337). We have designed the conformationally constrained antagonist analogs of bombesin by incorporating α,α-dialkylated amino acids. These antagonist analogs have shown anticancer activity in various cancers described in our U.S. patent application Ser. No. 09/630,633. In this application we have selected one of such antagonist analogs bombesin as component of the novel designed polypeptide.
Vasoactive intestinal peptide (VIP) is a widely distributed peptide hormone which mediates a variety of physiological responses including gastrointestinal secretion, relaxation of gastrointestinal, vascular and respiratory smooth muscle, lipolysis in adipocytes, pituitary hormone secretion, and excitation and hyperthermia after injection into the central nervous system. VIP receptors were characterized and localized in the neoplastic cells of most breast carcinomas, breast cancer metastases, ovarian adenocarcinoma, endometrial carcinomas, prostate cancer metastases, bladder carcinomas, colonic and pancreatic adenocarcinomas, gastrointestinal squamous cell carcinomas, non-small cell lung cancers, lymphomas, astrocytomas, glioblastomas and meningiomas (Reubi J C, (1995) J Nucl. Med., 36 (10):1846-53).
We have shown in our U.S. Pat. No. 6,156,725 that the eight residue VIP receptor binding inhibitor is antiproliferative to various cancers. In this patent application we have used this eight residue peptide as component for the novel designed polypeptide.
Substance P was the first gut neuropeptide discovered. It regulates gastrointestinal motility, increases blood flow in the gut, stimulates secretion of pancreas, salivary glands, small intestines and inhibits acid secretion. (Dockray, G. J., 1994,401 Gut peptides: Biochemistry and Physiology, Raven Press Ltd, New York). The role of Substance P in cancer has been well recognized particularly in small cell lung cancer. Several antagonists such as antagonist A, antagonist D and antagonist G of substance P have been described in literature which show inhibition of SCLC xenografts in vivo. (Wolf P. J. and Rozengurt, E., 1990, Can. Res. 50(13): 3968-73. Reeve, J. G. and Bleehen, N. M. 1994, Biochem. Biophys. Res. Commun. 199(3): 1313-19.)
We have designed the conformationally constrained antagonist analogs of substance P by incorporating α,α-dialkylated amino acids. These antagonist analogs have shown anticancer activity in various cancers described in our U.S. patent application Ser. No. 09/629,642. In this application we have selected one of such antagonist analog of substance P as component of the novel designed polypeptide.
The cyclic tetradecapeptide somatostatin was originally isolated from the hypothalamus and characterized as a physiological inhibitor of growth hormone release from the anterior pituitary. It was characterized by Guillemin et al. and is described in U.S. Pat. No. 3,904,594 (Sep. 9, 1975). Somatostatin has also been found to regulate insulin, glucagon and amylase secretion from the pancreas, and gastric acid release in the stomach. Its ability to inhibit the secretion of such hormones, allows somatostatin and its analogs to be therapeutically employed in clinical conditions for the treatment of acromegaly, pancreatic islet cell tumours, diabetes mellitus and gastrointestinal bleeding. Somatostatin receptors have been found to be over-expressed in a wide range of tumors, those arising in the brain (including meningioma, astrocytoma, neuroblastoma, hypophysial adenoma, paraganglioma, Merkel cell carcinoma, and gliomas), the digestive-pancreatic tract (including insulinoma, gluconoma, AUODoma, VIPoma, and colon carcinoma), lung, thyroid, mammary gland, prostate, lymphatic system (including both Hodgkin's and non-Hodgkin's lymphomas), and ovaries. It has been shown that somatostatin and some somatostatin analogues are capable of activating the tyrosine phosphatase enzyme, which antagonizes the effect of tyrosine kinases playing a very important role in the tumorous transformation [A. V. Schally: Cancer Res. 48, 6977 (1988)]. The importance of tyrosine kinases is supported by the fact that the majority of oncogenes code for tyrosine kinase and the major part of the growth factor receptors is tyrosine kinase [Yarden et al.: Ann. Rev. Biochem. 57, 443 (1989)].
We have designed the conformationally constrained agonist analogs consisting of eight amino acids of somatostatin by incorporating α,α-dialkylated amino acids. These agonist analogs have shown anticancer activity against various cancers and this is described in our U.S. Pat. No. 6,316,414, the subject matter of which is incorporated by reference. In this application we have selected one of such agonist analog of somatostatin as component of the novel designed polypeptide.
We have previously shown in our U.S. Pat. No. 6,156,725 and Australian patent No. 707,158; that Vasoactive intestinal peptide (VIP), Somatostatin, Substance P and Bombesin are secreted by some human tumor cells and that there are specific high affinity binding sites for these peptides on these cells. The four peptides were also shown to bind to tumor cells. The antagonist/analogs of these peptides were shown to have anti-proliferative activity on certain cancer cells, more specifically adenocarcinomas. A combination of the peptide antagonists/analogs was also shown to cause tumor regression in a nude mice xenograft model. It was hypothesized by us that there exists an autocrine mechanism for cell proliferation where the peptides are secreted by tumor cells and transduce cellular signals through specific cell surface receptors leading to cell proliferation. The analogs/antagonists to these peptides may then abrogate/block these cellular signals linked to proliferation. Further the antiangiogenic potential of the antagonist/analogs has also been described by us previously (U.S. application Ser. No. 09/248,381 and PCT application WO 00/047221).
We have also described in our U.S. Pat. No. 6,316,414 and U.S. patent application (Ser. Nos. 09/630,333; 09/630,345 & 09/629,642) (the subject matter of which is incorporated by reference) novel antiproliferative antagonist analogs of VIP, Bombesin, Substance P and agonist analogs of Somatostatin that are useful in the treatment of cancer. These analogs were designed incorporating α,α-dialkylated amino acids and show antiproliferative activity in a number of human tumor cell lines. Further, they caused partial tumor regression in nude mice xenografts. However, when used individually, these peptides had a narrow spectrum of activity with moderate levels of cytotoxicity. We have previously shown the synergistic in vivo tumor efficacy of these peptides in our U.S. patent application Ser. No. 09/896,903 in which it has been shown that when these peptides were used in combination as a mixture they exert a synergistic biological effect which is greater than the effect produced by any individual peptide alone.
In the present invention we have linked together the four designed peptides analogs of Somatostatin, Bombesin, Substance P and Vasoactive Intestinal Peptide, using a suitable linker, to form a single polypeptide. It is proposed that the individual peptides would be released from the polypeptide by appropriate enzymatic cleavage at the linker site in vivo.
Throughout the specification and claims, the following abbreviations are used with the following meanings:
BOP:Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexofluorophospatePyBOP:Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumHexofluorophospateHBTU:O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexofluoro-phosphateTBTU:2-(1H-Benzotriazole-lyl)-1,1,3,3-tetramethyluroniumtetrafluoroborateHOBt:1-Hydroxy BenzotriazoleDCC:Dicyclohexyl carbodiimideDIPCDI:Diisopropyl carbodiimideDIEA:Diisopropyl ethylamineDMF:Dimethyl formamideDCM:DichloromethaneNMP:N-Methyl-2-pyrrolidinoneTFA:Trifluoroacetic acid
In the formula (I) below and throughout the specification, the amino acids residues are designated by their standard abbreviations. Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by hyphen.
The following abbreviations are used for uncommon amino acids:                Orn=Ornithine        Pen=Penicillamine        Aib=α-Aminoisobutyric acid        Ac5c=1-Aminocyclopentane carboxylic acid        